ATP-dependent proteases degrade important regulatory proteins and help dispose of damaged and denatured proteins in the cell. Our research is focused on the specificity and mechanism of action of the Clp and Lon proteases of E. coli and human cells. Sequence analysis of a genomic clone of human LON, which produces an ATP-dependent protease targeted to mitochondria, has located the positions of 16 introns. This information will help in determining structurally distinct regions of Lon and suggests fusion joints for the construction of chimeric Lon proteases. To date, human LON cDNA clones, as well as several chimeras of human and E. coli lon, have proven to be unstable in E. coli, and the protein is poorly expressed and unstable. Other expression systems are being used (Vaccinia) or are under construction (yeast) to provide model systems to test the effects of mutational changes on Lon activity in vivo. In studies in which purified E. coli Lon protease was used to degrade purified CcdA, ATP hydrolysis was shown to be required for disruption of the secondary structure of CcdA, presumably to allow greater ease of entry to the proteolytic active sites. Conditions that stabilize secondary structure, such as functional interaction with other proteins, protect CcdA from degradation. The sequence of E. coli ClpA suggested, and electron micrographs confirmed, that the protein has two ATPase domains, and mutational studies indicated that the C-terminal domain was important for the activation of ClpP. In protease protection assays, addition of ClpP protected the C-terminal but not the N-terminal domain of ClpA, indicating that the C-terminal ATPase domain interacts with ClpP. A novel member of the Clp protease family, ClpYQ, was cloned and the proteins purified. ClpY and ClpQ were isolated separately and could be combined to form a high molecular weight complex with ATP-dependent protein degrading activity. Electron micrographs show that subunits of ClpQ are arranged in the form of a hexagonal ring. In this regard, ClpQ differs strikingly from the heptagonal 20 S proteasome, despite the significant degree of homology between these proteins. Binding of ATP stabilizes an oligomeric form of ClpY which also has a hexagonal structure. Because earlier studies had shown that ClpA is a hexamer and ClpP is a heptamer, it was postulated that asymmetric interactions between subunits may be important during the catalytic cycle. However, the identical six-fold symmetries of ClpY and ClpQ in the ClpYQ complex indicate that a symmetry mismatch is not essential for coupling activity of the ATP-dependent chaperone to that of the proteolytic component.